This invention relates to a method of field-flow fractionation (FFF), and more particularly to the use of relatively high field gradients to establish two-dimensional particle movement at the wall of a field-flow fractionation system.
FFF, disclosed previously in U.S. Pat. No. 3,449,938, is the descriptive term referring to a broad field of technology developed primarily for separation and characterization of macromolecules and particles. Generally, FFF has demonstrated a capability to deal with extreme ranges of mass, including particle sizes varying from a molecular weight of 600 to particles over 1 micrometer in diameter.
As explained in the referenced patent and also in a previous patent application of the present inventor (U.S. patent application Ser. No. 810,835), FFF involves the differentiation and segregation of particles along a flow channel under the influence of a force field applied across the flow channel. The effect of this field, which is usually applied perpendicular to the axis of the flow channel, is to force particles of different sizes into equilibrium layers of different effective thickness against a channel wall which operates as a restraining wall with respect to the particles. The thickness of the layers is determined primarily by (1) the interplay between the field-induced forces which tend to compact particles against the restraining wall and (2) Brownian motion which tends to disperse the particles away from the wall.
Steric field-flow fractionation or steric FFF was disclosed in U.S. patent application Ser. No. 953,655 and provides a method for separating particles according to size into discrete zones or into a continuous size spectrum in an effluent stream. It was suggested therein that the method was readily applicable to particles in the range 1-100 micrometers, and actual separations of considerable sharpness were carried out in the range 10-30 micrometers using samples of glass beads. Essentially, this method involved using a field strength of sufficient magnitude perpendicular to the direction of channel flow so that substantially all particles are pressed against one of the channel walls. In this sense, steric FFF is the limiting form of general field-flow fractionation (FFF) methodology. In steric FFF, the effective thickness of layers of particles forced by a field toward the wall of a flow channel is determined by particle size rather than by the normal processes of Brownian displacement. Since the influence of the former increases and the latter decreases with particle size, steric FFF is especially applicable to particles greater than 1 micrometer in diameter, whereas usually FFF is applied to submicron particles and macromolecules.
Steric FFF is therefore a method in which particles are rolled or tumbled along the smooth surface of a narrow channel by a flow stream confined in the channel. Large particles are tumbled more rapidly than small ones because they are forced by their own size further into the high velocity regions of the parabolic channel flow. The largest particles in the mixture therefore emerge first from the channel, followed by a continuum of decreasing sizes. It should be noted that this sequence is opposite to the normally observed elution of smaller to larger particles in FFF.
During fractionation under sedimentary steric FFF particles are held near the wall by gravity. The combination of the gravitational displacement toward the wall and the limitations on this displacement by the physical dimensions of the particle leads to well-defined and precisely positioned layers of particles of a given size, and it leads therefore to uniform displacement of the particles in the laminar flow stream of the channel.
Because particle displacement was confined to a single dimension in the previously described steric FFF, utility of the method was limited to fractionation of a single sample. Basically, such a sample was injected and the particles were allowed to separate prior to further sample injection. Such a system was not suitable for continuous operation in view of the differential rate of particle displacement along one axis only. This displacement would result in migration of larger particles into preceding groups of smaller particles, if multiple or continuous sample injections occurred. Therefore, FFF has heretofore continued under the limitation of single sample injection or batch operation, without a method for continuous operation.